The K562 erythroleukemia cell Ene constitutively expresses low levels of embryonic and fetal but not adult hemoglobin. Hemoglobin production can be further induced by chemical stimuli such as hemin. An in vitro transcription assay based on the soluble cell-free nuclear extracts obtained from K562 cells has been used to investigate the regulation of human globin genes expression at a transcriptional level. To evaluate the regulatory role of 5' upstream cis-acting sequences in epsilon-globin gene transcription, six deletion mutations on 5' upstream to the cap site (del- 65,del-104,del-177, del-274, del-453, del-535) were constructed and were examined by an in vitro transcription assay. To detect a signal of correctly initiated transcription from the cap site, the resultant run-off transcripts were hybridized with (32)P labeled antisense RNA probe followed by ribonuclease T1 digestion (T1 analysis). The T1 analysis of the deletion mutations demonstrated negative and positive transcriptional regulatory cis-acting elements in proximal promoter region of epsilon-globin gene. In order to identify the transacting factors related to these positive and negative regulatory cis-acting elements of epsilon-globin gene, we fractionated K562 nuclear extracts by means of an anion exchange chromatography with step-wise ammonium sulfate concentration elution were employed to drive transcription from each deleted promoter of six mutations. The 175 mM fraction (F175) which was previously reported as the most transcriptionally active fraction showed no differential expression among the deletion mutations, suggesting the absence of specific protein factors which may interact with the negative cis-acting elements in the F175. The beta-globin gene could not transcribed in vitro by K562 nuclear extracts; however, the combination extracts of K562 nuclear extract and high-speed cytoplasmic extract of K562 cells (K562 S 100) directed the transcription from B-globin gene promoter. In order to confirm that the signal is an accurately initiated transcription, antisense RNA probe was made and the T1 analysis was carried out. The supplement of K562 nuclear extract with K562 S 100 did induce the correct initiated transcription from the cap site of the beta-globin gene.